Klebsiella michiganensis: a nitrogen-fixing endohyphal bacterium from Ustilago maydis

Area of Science:

• Mycology
• Microbiology
• Plant Pathology

Background:

• Ustilago maydis causes significant corn smut disease.
• Intracellular bacteria within pathogenic fungi are poorly understood.
• Investigating fungal-bacterial endosymbiosis offers insights into host-pathogen interactions.

Purpose of the Study:

• To isolate and identify bacteria residing within Ustilago maydis hyphae.
• To characterize the symbiotic relationship and functional capabilities of the endohyphal bacterium.
• To assess the potential of the bacterium for nitrogen fixation.

Main Methods:

• Isolation of Ustilago maydis strain YZZF202006 from infected corn.
• Confirmation of intracellular bacteria using PCR amplification and fluorescence in situ hybridization (FISH).
• Genomic analysis (Average Nucleotide Identity - ANI) and phylogenetic analysis for bacterial identification.
• Culturing, characterization, GFP-labeling, and reintroduction of the bacterium into U. maydis.
• Acetylene reduction assay to quantify nitrogenase activity.
• Genome sequencing to identify nitrogen fixation genes.

Main Results:

• An endohyphal bacterium, later identified as Klebsiella michiganensis YZUMF202001, was isolated from U. maydis.
• K. michiganensis was successfully reintroduced and confirmed to live within U. maydis hyphae.
• The bacterium exhibited significant nitrogenase activity (646.25 ± 38.61 nmol·mL⁻¹·h⁻¹ C₂H₄) and could grow on nitrogen-free media.
• A complete cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was identified in its genome.

Conclusions:

• Klebsiella michiganensis is an endohyphal bacterium found within Ustilago maydis.
• This endosymbiont possesses functional nitrogen fixation capabilities.
• The endohyphal K. michiganensis may contribute to nitrogen fixation for the U. maydis fungus, offering potential applications in agriculture.